目的:构建靶向人钠氢交换蛋白-1(NHE-1)RNA干扰(RNAi)腺病毒表达载体,感染SH-SY5Y细胞,建立细胞内酸化模型,为进一步研究细胞内pH值变化与散发性阿尔茨海默病间的关系奠定基础.方法:利用基因重组技术构建4个含NHE-1前体微小RNA(miRNA)的重组干扰质粒pcDNA(TM)6.2-GW/miR和阴性对照质粒DcDNA(TM)6.2-GWeg-miR,实时荧光定量逆转录-聚合酶链反应(RT-PCR)检测NHE-1 mRNA表达变化,筛选最佳干扰靶序列;BP和LR重组系统获得腺病毒干扰质粒pAd-miR-NHE-1,经包装、纯化后检测腺病毒滴度;感染复数(MOI)值梯度试验确定靶向人NHE-1 RNAi腺病毒感染SH-SY5Y细胞最佳MOI值,以最佳条件感染SH-SY5Y细胞,荧光探针法检测不同处理组细胞内pH值.结果:聚合酶链反应(PCR)提示最佳靶向人NHE-1 RNAi病毒表达载体构建成功,且能有效抑制NHE-1 mRNA表达;靶向人NHE-1 RNAi腺病毒感染SH-SY5Y细胞最佳MOI值为160.当最佳MOI值为160时,NHE-1 miRNA腺病毒感染24、48和72h后SH-SY5Y细胞内pH值分别为5.97±0.03、5.98±0.02和5.98±0.02;空载体对照组为6.05±0.04、6.04±0.07和6.03±0.05;空白对照组为6.03±0.06、6.05±0.04和6.03±0.04.不同测量时间点,SH-SY5Y细胞内pH值均显著低于空载体对照组和空白对照组,差异具有统计学意义(24h:P=0.002.0.015;48h:P=0.030,0.012;72h:P=0.018,0.010);但感染RNAi腺病毒后不同测量时间点(24、48和72h)SH-SY5Y细胞内pH值差异元统计学意义(P=0.762).结论:最佳靶向人NHE-1 RNAi腺病毒表达载体及细胞内酸化模型的成功建立,可为探索细胞内酸碱平衡与β-淀粉样蛋白产生之间的关系提供一种有效工具.%Objective To construct adenovirus - mediated RNA interference (RNAi) expression vector of targeting human Na+-H+ exchanger-1 (NHE-1) gene to infect SH-SY5Y cells and establish intracellular acidification model in order to lay the foundation for researching the relation between intracellular pH value and sporadic Alzheimer's disease (AD).Methods Four kinds of recombined interfering plasmid pcDNA? 6.2 - GW/miR including specific pre - miRNA single stranded DNA oligonucleotide for NHE-1 and negative control plasmid pcDNA? 6.2-GWeg-miR were constructed.The recombined plasmid with best interfere efficiency was detected by real-time fluorescent quantitative reverse transcriptase polymerase chain reaction (RT-PCR) technique.The best interfere fragment was recombined with pDONR221 vector and pAD/CMV/V5-DEST-GFP vector in turn by BP recombination system and LR recombination system.Recombinant adenovirus pAd - miR - NHE - 1 was identified, purified, proliferated and titrated.The best multiplicity of infection (MOI) was determined by MOI gradient test.With optimum condition transfected SH - SY5Y, the intracellular pH was detected by fluorescent probe.Results PCR showed that the best RNAi expression vector of targeting human NHE-1 gene was constructed successfully.It could inhibit NHE-1 mRNA expression successfully.The best MOI was 160.According to the optimized MOI, SH-SY5Y cell line was infected by NHE-1 miRNA adenovirus.After 24, 48 and 72 h, the intracellular pH of adenovirus transfect group was 5.97 ± 0.03, 5.98 ± 0.02 and 5.98 ± 0.02, respectively; the intracellular pH of empty vector transfect group was 6.05 ± 0.04, 6.04 ± 0.07 and 6.03 ± 0.05, respectively; the intracellular pH of blank control group was 6.03 ± 0.06, 6.05 ± 0.04 and 6.03 ± 0.04, respectively.After 28, 48 and 72 h, the intracellular pH of adenovirus transfect group was decreased significantly compared to empty vector transfect group and blank control group (24 h: P = 0.002, 0.015; 48 h: P = 0.030, 0.012; 72 h: P = 0.018, 0.010).But at different period after transfection, the intracellular pH was of no obvious difference (P = 0.762).Conclusion The best RNAi expression vector of targeting human NHE - 1 gene is successfully constructed and the intracellular acidification model is established which can provide an effective tool for further study of the relation between intracellular acid base equilibrium and P-amyloid (AP) production.
展开▼
